Specific CD4+ T cell phenotypes associate with bacterial control in people who ‘resist’ infection with Mycobacterium tuberculosis

A subset of individuals exposed to Mycobacterium tuberculosis (Mtb) that we refer to as ‘resisters’ (RSTR) show evidence of IFN-γ− T cell responses to Mtb-specific antigens despite serially negative results on clinical testing. Here we found that Mtb-specific T cells in RSTR were clonally expanded, confirming the priming of adaptive immune responses following Mtb exposure. RSTR CD4+ T cells showed enrichment of TH17 and regulatory T cell-like functional programs compared to Mtb-specific T cells from individuals with latent Mtb infection. Using public datasets, we showed that these TH17 cell-like functional programs were associated with lack of progression to active tuberculosis among South African adolescents with latent Mtb infection and with bacterial control in nonhuman primates. Our findings suggested that RSTR may successfully control Mtb following exposure and immune priming and established a set of T cell biomarkers to facilitate further study of this clinical phenotype.

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Introduction:
1.You mention other cohorts of 'resisters' but these are all quite different from your long-term resister cohort.I would suggest differentiating between 'early clearance' and resistance mechanisms as they are likely to involve different cell types.2.You mention the SA cohort were long-term non-progressors but they were non-progressors from disease, not infection.This needs to be clarified as it's very likely different mechanisms will be at play.

Methods:
1. How did you determine the community controls were low exposure?2. Why did you focus solely on antigen-specific T cells and not B cells etc? 3. Why did you use peptide pools rather than WCL for eg.Or Mtb300 -this would allow a much broader response to be analysed.

Results:
1.In all these studies, analysis is done only after classification is known.It would be good to know if there is prognostic risk for development of LTBI as opposed to simply analyzing the differences once converted.It would be good to determine the cell types in individuals at baseline time-point ie in those who convert and those who don't convert prior to any evidence of infection.This would indicate if the same cell types are dominant in RSTR at future time-points.2. For the HIV infected individuals, were they on ART?What were their CD4 counts and what were their QFT and TST readings?3. Do you think protection from disease progression (ie the ACS cohort) would require the same cell types?Given progressors are infected then I would hypothesise that different mechanisms are required, and likely involve both IFN-dependent and independent responses.4. Suggest looking at ratio of Treg to other cell types as it's normally the overall response with a balance of pro and anti-inflammatory that determines outcome. 5. Could the transcriptional profiles have changed following sorting (and thus activation)?6.You say cluster 6 'appears to be' enriched.Please be more precise as to whether it was or not… 7. Interesting results for RSTR in terms of clonal expansion.Perhaps this lack of skewing to a certain type of response may aid protection depending on which antigen is presented?8. Results for higher levels of naïve phenotype in RSTR are not significant, particularly if you were to adjust for multiple comparisons.Please change wording to 'shows a trend'.9. Which time-points were analyzed for luminex?Could these same time-points be analysed using PBMC in future studies?Note, I am not suggesting further analysis for this manuscript.10.The NHP data are very interesting.What about data from iv BCG? Do those animals who were protected show similar gene profiles?That data should be publicly available.

Discussion:
This was really short and perhaps didn't provide enough detail on the limitations of each model.The South African cohort were not resistant to infection and whilst the data provides interesting insight into protection from disease, it's not necessarily the same mechanisms/cell types at play.In addition, adolescents will be closer to the time of BCG vaccination and likely will have some influence on protection from infection due to vaccine status.I think the RSTR cohort is unique in that it is very long term after exposure and therefore would benefit from more discussion on this long-term protection versus protection at early stages of exposure versus protection from disease.In addition, the obvious omission in human studies is lack of analysis at the site of infection and this limitation should also be discussed in relation to the NHP findings.
Methods: 1.Why were different staining methods used for the endemic controls and the HHC? Are the results generated therefore comparable? 2. Please explain why no multiple comparison corrections were used?Reviewer #2: Remarks to the Author: Sun et al set out to understand early events of Mtb infection with the main question been if resisters have any unique anti-bacterial functional programs relative to latently infected persons.A long outstanding question in the field of TB is understanding the phenomenon of resistors and whether failure to convert TST or IGRA has a biological association with Mtb clearance.They aimed to measure IFNg-independent T cell signatures specific to household exposure, and to determine genes selectively enriched among Mtb-specific T cells derived from resisters compared to controls.This will fill in knowledge gaps about whether resisters have actual protective immunity or failed to convert TST/IGRAs in addition to the known Epi data.They observed a role of Th17 cells and lower polyfunctionality scores in TST-/IGRA relative to LTBI, in addition, they showed important information on T cell quality which is novel.They used other cohorts to confirm this and non-human primates.The manuscript is well-written, experiments were well described as well as the results and conclusions which were precise and concise.The study is important and would have a major impact on the field, however, some of the claims are not confirmed resulting in several limitations.I'm requesting for a major revision due to importance of the paper in the field.

Major limitations
The study is important and will have a major impact on the field, however these points should be addressed before the paper can be considered for publication in Nature Medicine.
• The authors claim that the observed Th17 phenotype (a known correlate of protection both in human and animal studies); they show is reflective of bacterial burden.However, they have not proven this.I would recommend they measure bacterial burden in the study cohorts to confirm this claim.How do the bacterial burden correlate with the responses observed?
• TB has a diverse set of antigens, the authors used only two antigens, ESAT-6 and CFP which are used in IGRAs.The study participants were classified based on IGRAs and TSTs, and persons who were IGRA negative were classified as resistors.IGRAs measure IFNg responses from T cells, thus their observed results that the quality of IFNg response in resistors is low is expected.Is the response observed after ESAT^ and CFP10 stimulation the same as in other TB antigens such as MTB300?I'm recommending that the authors test other TB antigens in the same way as ESAT-6/CFP10 to confirm their results.
• The resistor/LTBI cohort used for targeted sequencing is small.Sample size should be increased to increase the statistical observations made in Figure 2 after index sorting and targeted sequencing.
• SELECT-Seq was used to characterize responses observed in the targeted sequencing, cells were sorted on TCRab and activation induced markers.I recommend showing data from the TCR observed in the study to provide more information on the diversity of the T cells involved in protection.
• The authors claim that the T cells from the resistors are more stem cell naïve-like based on expression of a few stem naïve genes.This has not been functionally validated and should be validate via flow using naïve like stem cell markers.
• There are no mechanistic studies which are important in comprehending the underlying mechanisms for T cell protection in TB disease.Proliferative responses of clonally expanded cells are needed to show functional ability of the cells after stimulation.

Minor comment
• The study was validated in a cohort of adolescents based on gene expression data, which is excellent.Cellular data is also available from the same cohort (Scriba et al., 2017, Plos Pathogens)  and this should be analyzed to confirm the T cell responses observed in the resistors. [REDACTED]

Point-by-Point Response to Reviewer Comments
Reviewer #1: This study analyzed individuals with a high probability of exposure to M. tuberculosis (M.tb) who appear to 'resist' infection.While these 'resisters' (RSTR) display IFN-γ-independent T cell responses to the M.tb-specific antigens ESAT-6 and CFP-10, it is currently unknown whether specific T cell functional programs are associated with this clinical outcome.The data presented outlines results from 3 different human cohorts and a NHP cohort to determine genes and cell pathways involved in this 'protection'.The authors found that M.tb-specific T cells derived from RSTRs showed an early differentiation phenotype as well as enrichment of Th17-like transcriptional programs compared to LTBIs, which were characterized by Th1*-like effector programs.The paper was interesting, very well written and the data presented built on nicely from previous research from this group.The inclusion of community controls, and comparison with other models, provides valuable information on potential mechanisms of protection in this unique cohort.
We appreciate this positive assessment of our work.
Outlined below are some minor concerns/points for clarity: Introduction: 1.You mention other cohorts of 'resisters' but these are all quite different from your long-term resister cohort.I would suggest differentiating between 'early clearance' and resistance mechanisms as they are likely to involve different cell types.This is an excellent suggestion.We have revised the Introduction to more clearly communicate the differences among these cohorts.
Lines 81-85: Other studies have also reported highly exposed individuals who demonstrate negative TST and/or IGRA among healthcare workers, miners, and household contacts 8 .The consistency of these reports is striking despite variations in timing and strength of exposure, duration of follow-up, and frequency of testing 7,9 .Whether these individuals have controlled M.tb infection is unknown.
Lines 91-92: However, T cells likely play a role in mediating 'resistance' to established M.tb infection, in particular in long-term 'resistance' to established M.tb infection 11,12 .Lines 424-427: Though our experiments focused on characterizing T cell phenotypes in long-term resistance, our results are not inconsistent with a model in which M.tb is recognized and eliminated without the assistance of T cells at early stages of exposure and protection from disease.
2. You mention the SA cohort were long-term non-progressors but they were non-progressors from disease, not infection.This needs to be clarified as it's very likely different mechanisms will be at play.In response to this comment, we have extensively revised the manuscript to reflect the associations between T cell phenotypes and the full spectrum of human tuberculosis, ranging from 'resistance' to infection and non-However, the current work is meant to directly follow up our original study which reported IFN-γ-independent T cell responses in both RSTR and LTBI subjects (PMID: 31110348).We have added the following text in the Introduction to clarify this: Lines 113-118: Consistent with this literature, we have previously shown that nearly all Ugandan household contacts with a high probability of M.tb exposure display IFN-γ independent T cell responses to M.tb-specific antigens, including IL-2, TNF, and CD154 7 .In a study of South African miners, we also showed that RSTRs harbored M.tb-specific antibodies with unique Fc receptor profiles 26 .However, it is currently unknown whether M.tb-specific T cells from RSTRs harbor any unique anti-bacterial T cell functional programs compared to LTBI controls.
5. Why did you use peptide pools rather than WCL for e.g.Or Mtb300 -this would allow a much broader response to be analysed.We initially focused on characterizing T cell responses to ESAT-6 and CFP-10 because those antigens form the bedrock of clinical TB diagnosis and are incorporated into multiple commercial assays.However, in response to this critique as well as that of Reviewers 2 and 3, we generated new data using M.tb whole cell lysate, which contains many more antigens including several that are not specific for M.tb.Specifically, we conducted a follow up experiment in which we analyzed a discovery cohort of RSTRs (n=16) and LTBIs (n=16) using index sorting and targeted transcriptional profiling followed by a validation cohort of RSTRs (n=17) and LTBIs (n=20) using flow cytometry and Luminex after whole M.tb lysate stimulation (see revised Fig 1a study schema below).Overall, we did not observe qualitative differences in the transcriptional profiles of CD4 T cells between the two groups.However, we did replicate the association between Th17-like and T-regulatory T cells and RSTRs seen with ESAT-6/CFP-10 stimulation.The data have been incorporated into new Figure 6 and associated Results text, which are copied below in full.Having established the relative enrichment of several ESAT-6/CFP-10 specific T cell phenotypes among RSTRs compared to LTBIs, we next sought to determine whether the same phenotypes were enriched after stimulation with M.tb whole cell lysate, which contains a broader set of antigens that are conserved across mycobacteria.Compared to experiments with ESAT-6/CFP-10 stimulation, we examined a larger discovery cohort of RSTRs (n = 16) and LTBIs (n = 16) using index sorting and targeted transcriptional profiling.Overall, we did not observe qualitative differences in CD4 T cell phenotypes between the two groups (Supplementary Fig. 7a).Nevertheless, we attempted to replicate our original findings in an independent set of samples (RSTRs n = 17, LTBI n = 20) with flow cytometry and Luminex (Fig. 1A, Supplementary Fig. 8, and Table 2).T cells responsive to M.tb lysate stimulation that expressed an early differentiation phenotype were present at similar frequencies in RSTRs and LTBIs (Supplementary Fig. 6b).However, RORγt+T-bet+ T cells were present at higher frequencies among LTBI subjects and RORγt+T-bet-T cells were higher among RSTRs, analogous to what we observed with ESAT-6/CFP-10 stimulation (p=0.039 and p=0.011, respectively) (Fig. 6a and 6b).These cells were also characterized by co-expression of CCR6 and CXCR3, supporting their designation as Th1* T cells (Fig. 6c).As expected, IFN-γ production after M.tb lysate stimulation was higher among LTBI subjects when measured by intracellular cytokine staining or Luminex (p < 0.001 and p < 0.001, respectively) (Fig. 6d).IL-17Aexpressing CD4 T cells also trended higher in RSTRs; however, this was not statistically significant (Fig. 6e and  6f).Notably, the Th17-promoting cytokine IL-23 was significantly higher in conditioned supernatants from RSTRs compared to LTBIs (p=0.041) (Fig. 6g).
We next examined the frequency of M.tb lysate-responsive regulatory T cells as defined by the expression of CD25 or co-expression of FoxP3 and CD25.We observed a higher frequency of both CD25+ and FoxP3+CD25+ T cells after M.tb lysate among RSTRs compared to LTBIs (p=0.052 and p=0.013, respectively) (Fig. 6h, 6i, and 6j).In addition, IL-10 concentrations in conditioned supernatants trended higher, though this was not statistically significant (Fig. 6k).The ratio of the anti-inflammatory regulatory T cell fraction to the proinflammatory T cell fraction, including Th1, Th17, and Th1* cells, was significantly higher in RSTRs compared to LTBIs (Supplementary Fig. 7c).Taken together, these data show that enrichment of Th17-like and T-regulatory functional programs observed among RSTRs after stimulation with ESAT-6 and CFP-10 are also recapitulated with a broader set of antigens present in M.tb lysate.
Results: 6.In all these studies, analysis is done only after classification is known.It would be good to know if there is prognostic risk for development of LTBI as opposed to simply analyzing the differences once converted.It would be good to determine the cell types in individuals at baseline time-point i.e. in those who convert and those who don't convert prior to any evidence of infection.This would indicate if the same cell types are dominant in RSTR at future time-points.
We appreciate this comment, and addressing this question is indeed the focus of current work which requires enrolling a completely independent cohort and is beyond the scope of the current manuscript.Dr. Boom is leading those efforts as PI of our IMPAc-TB Consortium but we will not have results for several years.Nevertheless, the current study is still extremely valuable for the field as acknowledged by this Reviewer because it builds on a cohort that was established and followed for nearly 10 years with clear clinical outcomes.
7. For the HIV infected individuals, were they on ART?What were their CD4 counts and what were their QFT and TST readings?
We apologize for the confusion.There were no HIV-infected persons included in our study.We have clarified this in the Methods.
Line 502: We were able to classify 196 HIV-uninfected individuals based on TST and QFT concordance.
Lines 522-524: PBMC from a subset of these definite LTBI controls and RSTRs were used for the present study and selected after matching for age, sex, exposure risk score, and documented lack of HIV co-infection.
8. Do you think protection from disease progression (i.e. the ACS cohort) would require the same cell types?Given progressors are infected then I would hypothesise that different mechanisms are required, and likely involve both IFN-dependent and independent responses.
We agree with this comment as well as that from R3 and feel that we can do a better job of comparing and contrasting the associations we report in the two human cohorts.We do acknowledge the difference but want to address the similarity in the IFN-γ-independent response among different cohorts.
We have edited the Discussion section as follows: Lines 446-456: There is emerging evidence that IFN-γ dependent T-cell immunity in humans may be a reliable proxy for established infection with M.tb.IFN-γ concentrations derived from IGRAs are associated with progression to active TB 70,71 .In addition, there is a progressive increase in M.tb-specific IFN-γ dependent CD4 T cell responses across the spectrum of IGRA non-converters, reverters, and persistent IGRA+ South African adolescents 72 .The clinical relevance of IFN-γ independent T-cell profiles has not been thoroughly explored.Our data suggest that IL-17 or IL-23 production in the absence of IFN-γ after stimulation with ESAT-6/CFP-10 may also have clinical utility.Expanded analysis of IGRA supernatants has already revealed host biomarkers that distinguish latent from active TB 73 .As IL-17, IL-21, and IL-23 concentrations are expected to be very low after peptide stimulation, more sensitive assays may be required to realize their full diagnostic potential.
Lines 482-489: Surprisingly, we also found an expansion of this Th17-like transcriptional program among a subset of South African adolescents with established LTBI that fail to progress to active TB relative to progressors.Thus, it appears that Th17-like T cells may mediate protection across the spectrum of M.tb infection and disease and across species.At a minimum, it supports the notion that the current definition of 'LTBI' is heterogeneous and consists of individuals at risk and protected from M.tb disease.Future work may eventually render the terms 'RSTR' and 'LTBI' obsolete because they are defined by IFN-γ dependent immunity and replace them with Th17-based, more clinically informative definitions.9. Suggest looking at ratio of Treg to other cell types as it's normally the overall response with a balance of pro and anti-inflammatory that determines outcome.This is an excellent suggestion.We performed the analysis as suggested by the reviewer.Among M.tb-specific T cells, we found that the ratio of Treg to Th1 and Th17 cells was higher among RSTRs than LTBI subjects These data have been incorporated into a new Supplementary Figure 7 and we have added the following text to the Results and Discussion: In the Supplementary Figures PDF: Supplementary Lines 345-349: In addition, IL-10 concentrations in conditioned supernatants trended higher, though this was not statistically significant (Fig. 6k).The ratio of the anti-inflammatory regulatory T cell fraction to the proinflammatory T cell fraction, including Th1, Th17, and Th1* cells, was significantly higher in RSTRs compared to LTBIs (Supplementary Fig. 7c).
10. Could the transcriptional profiles have changed following sorting (and thus activation)?
We appreciate this point but note that we and others have successfully employed this method to successfully identify rare antigen-specific T cells in previously published work (PMID: 32341563, 30992377, 36604540).Per the Methods, the stimulation time was six hours while the time required for sorting was less than 1 hour.Cells were directly sorted into lysis buffers to capture their functional profiles without additional manipulation.We have added the following text to the Methods to clarify this point: Lines 547-549: We conducted the SELECT-seq protocol, which includes stimulation and sorting procedures, as described in several recent publications 31,55,79 .In brief, PBMCs from RSTR and LTBI samples were stimulated as described in the ICS methods above.
11.You say cluster 6 'appears to be' enriched.Please be more precise as to whether it was or not… We apologize for the confusion.We have clarified this point in the Results.
12. Interesting results for RSTR in terms of clonal expansion.Perhaps this lack of skewing to a certain type of response may aid protection depending on which antigen is presented?Indeed, this is one possible hypothesis and the topic for future work.We have expanded upon this point in the Discussion.
Line 458-468: Our initial experimental approach was focused on defining the functions of T cells targeting ESAT-6 and CFP-10 because these are specific for M.tb and incorporated into IGRAs.We extended our approach to include M.tb lysate, which contains many more antigens, but at the expense of specificity for M.tb.Nevertheless, we found concordant results when examining Th17-like and T-regulatory T cell phenotypes.In a study comparing M.tb-infected mice and BCG-vaccinated humans, M.tb infection drove ESAT-6 specific T cells to become more differentiated that Ag85B-specific T cells 74 .This was consistent with the observation that M.tb restricts expression of Ag85B but not ESAT-6 during chronic infection 75,76 .Musvosvi et al. also recently reported preferential targeting of PE13 and CFP-10 by non-progressors compared to progressors 55 .Similarly, RSTRs may preferentially target certain mycobacterial antigens when compared to LTBIs, which will be the subject of future work.
13. Results for higher levels of naïve phenotype in RSTR are not significant, particularly if you were to adjust for multiple comparisons.Please change wording to 'shows a trend'.
We made the change as requested to the text.In addition, we generated new data to demonstrate the functionality of stem cell memory T cells in both RSTRs and LTBIs (see response to Reviewer #2, Comment #5 below).
14. Which time-points were analyzed for Luminex?Could these same time-points be analysed using PBMC in future studies?Note, I am not suggesting further analysis for this manuscript.
We apologize for the confusion.We have clarified the choice of these time points in the Methods and highlight the specific ones displayed in the figure legends (Figure 4J-K and Figure 5J).
Lines 691-699: Background-corrected signals were computed by subtracting the signal from the DMSO condition from the antigen stimulation condition.To choose the precise time points with which to compare protein secretion (measured by Luminex and ELISA) between the RSTR and LTBI groups, we identified samples exhibiting the most favorable signal-to-noise ratio in response to stimulation (ESAT-6/CFP-10 or M.tb lysate) versus DMSO.This selection was based on applying the T-statistic at each time point assessed.Subsequently, statistical testing between the two groups was performed on a single time point for each analyte using the Students' t-test (Supplementary Fig. 6 and Supplementary Fig. 8).
15.The NHP data are very interesting.What about data from iv BCG? Do those animals who were protected show similar gene profiles?That data should be publicly available.To directly address this critique, we collaborated with Yiran Liu and Purvesh Khatri (lead authors of PMID: 37390827) to examine whether T cell phenotypes that we observed as associated with RSTR status were also associated with protection after IV BCG.Indeed, we found that a Th17 summary score derived from data reported here was also associated with protection (revised Fig 7i).We also collaborated with Dr. Changqi Wang and Dr. Doug Lauffenberger (authors of PMID: 37267955) to examine whether IFN-γ independent CD4 T cell profiles were associated with protection in the Darrah et al. study.Indeed, we found that CD4 T cells producing IL-17 but not IFN-γ, TNF, or IL-2 were associated with protection after IV BCG vaccination.These data substantially strengthen the original claims in our paper.We have revised Figure 7 4.The score was computed as the geometric mean of gene expressions plotted for protected and non-protected groups according to the total CFU M.tb upon necropsy.Statistical testing was performed using the Wilcoxon rank-sum test.(i) Flow cytometry data from a parallel study examining airway T cells induced by IV BCG vaccination in rhesus macaques 58 was used to calculate the cell count and the cell fraction of Th17 cells defined as IFNγ-IL2-IL17+TNF-CD4 subset in BALs.The violin plots depict the difference between the protected and non-protected groups according to the total CFU M.tb upon necropsy.Statistical testing was performed using the Wilcoxon rank-sum test.
Lines 394-402: The associated gene module scores were significantly higher in RSTRs compared to LTBIs (Fig. 7g).Next, we examined blood transcriptomes and T cell phenotypes in rhesus macaques that were vaccinated with intravenous BCG and subsequently challenged with M.tb in an effort to identify correlates of protective immunity 57,58 .Expression of a Th17 gene module based on genes enriched in T cells from RSTRs was higher among macaques that were protected against M.tb challenge compared to those that were not protected (Fig. 7h).Similarly, the absolute number but not the frequency of purified protein derivative (PPD)-specific IFN-γ independent IL-17+ CD4 T cells in bronchoalveolar lavage was higher among protected macaques (Fig. 7i).

Non-human primate cohort analysis
We derived the gene expression patterns identified in 26 granulomas from four cynomolgus macaques obtained ten weeks after low-dose M.tb infection and analyzed using single-cell RNA sequencing 56 .We selected the top 15 enriched genes in each of the stem-like cell subsets and T1-T17 population 1 cell subset and calculated their mean expression in RSTR and LTBI donors using the SELECT-seq dataset.Genes that were not expressed or not found were excluded from the analysis.We used the AddModuleScore function from Seurat 86 to calculate the associated gene module scores.
We examined the expression of the Th17 gene module in the whole blood transcriptome of 34 rhesus macaques from a dose-ranging study of intravenous (IV) BCG vaccination followed by M.tb challenge.Data collection and pre-processing have been described in detail previously 57 .We calculated expression summary scores using the geometric mean of the Th17 module genes identified in Fig. 4. We then compared summary scores across all timepoints between macaques that were protected (n = 18) versus not protected (n = 16) against M.tb challenge, as determined by total M.tb CFU upon necropsy, using the Wilcoxon rank-sum test.Flow cytometry was performed on freshly collected bronchoalveolar lavage fluid obtained from these same macaques and detailed methods have been previously published 58 .From these data, we extracted the IL17-monofunctional T cell phenotype measured by IFNγ-IL2-IL17+TNF-among CD4 T cells after PPD stimulation.Discussion: 16.This was really short and perhaps didn't provide enough detail on the limitations of each model.The South African cohort were not resistant to infection and whilst the data provides interesting insight into protection from disease, it's not necessarily the same mechanisms/cell types at play.In addition, adolescents will be closer to the time of BCG vaccination and likely will have some influence on protection from infection due to vaccine status.
We agree with this point.We have expanded this point in the Discussion and include both strengths and potential limitations in making this connection.
Lines 424-428: Though our experiments focused on characterizing T cell phenotypes in long-term resistance, our results are not inconsistent with a model in which M.tb is recognized and eliminated without the assistance of T cells at early stages of exposure and protection from disease.Alveolar macrophages are among the first airway immune cells to encounter M.tb and are generally permissive to M.tb growth 59, 60 … Lines 446-450: There is emerging evidence that IFN-γ dependent T-cell immunity in humans may be a reliable proxy for established infection with M.tb.IFN-γ concentrations derived from IGRAs are associated with progression to active TB 70,71 .In addition, there is a progressive increase in M.tb-specific IFN-γ dependent CD4 T cell responses across the spectrum of IGRA non-converters, reverters, and persistent IGRA+ South African adolescents 72 .
Lines 480-487: We report a higher frequency of RORγt+T-bet-M.tb-specificT cells among RSTRs compared to household contacts with established LTBI in Uganda.Surprisingly, we also found an expansion of this Th17-like transcriptional program among a subset of South African adolescents with established LTBI that fail to progress to active TB relative to progressors.Thus, it appears that Th17-like T cells may mediate protection across the spectrum of M.tb infection and disease and across species.At a minimum, it supports the notion that the current definition of 'LTBI' is heterogeneous and consists of individuals at risk and protected from M.tb disease.17.I think the RSTR cohort is unique in that it is very long term after exposure and therefore would benefit from more discussion on this long-term protection versus protection at early stages of exposure versus protection from disease.In addition, the obvious omission in human studies is lack of analysis at the site of infection and this limitation should also be discussed in relation to the NHP findings.
We agree with these points and have revised the Discussion to address them as follows: Line 428-444: Studies of blood-derived myeloid cells have revealed differences in the transcriptional response between RSTRs and LTBIs, which we have reported in this cohort as well as in a parallel cohort of gold miners in South Africa 61 …These mechanisms may act in concert to reduce the bacterial/antigen load and tune the inflammatory environment to prime the T cell phenotypes that we observed.Further investigation of M.tb-specific T cells in the lungs of RSTRs will be required to confirm the findings we report here using peripheral blood 69 .

Methods:
18. Why were different staining methods used for the endemic controls and the HHC? Are the results generated therefore comparable?
We apologize for the confusion.In the interest of transparency, we sought to highlight minor methodologic differences between staining methods used in the two cohorts.Rather than distract the reader, we have elected to consolidate the text.
Lines 537-544: Intracellular cytokine staining (ICS) was performed on samples from the endemic controls as previously described 7 .The same ICS assay and flow cytometry acquisition method were performed on samples from the household contacts with minor modifications.Prior to staining, samples from the household contacts were divided in half to be analyzed using two multiparameter flow cytometry panels, one for the analysis of Tregs and one for Th subsets (Supplementary Table 2).Cells were permeabilized and underwent intracellular staining using the eBioscience Foxp3/Transcription Factor Staining Buffer Set (eBioscience, San Diego, CA) to allow for the analysis of transcription factors.

Please explain why no multiple comparison corrections were used?
We apologize for the confusion.Broadly speaking, we designed our study with a 'discovery' and 'validation' phase from the outset.The discovery phase was meant to be hypothesis generating with a high tolerance for false discovery.We identified key hypotheses from the discovery data (targeted transcriptional profiling and SELECT-Seq) based not only on statistical significance but biological rationale to follow up in the validation phase using independent methods (flow cytometry and Luminex) and non-overlapping samples (Fig. 1 schema).We also performed targeted testing of our key hypotheses in external data sets using data derived from both human and non-human primates.(Fig. 7).Thus, our overall approach provides several layers of experimental and biological validation without focusing only on statistical significance.Where multiple features were measured and analyzed simultaneously, we did correct for multiple hypothesis testing using the Bonferonni Method, and this is indicated in the Fig legend (Fig. 1d, 3d, 4b, 5c, etc).We have made the following changes to the Figure legends to make this point more explicitly: Lines 665-667: We used Wilcoxon rank-sum tests to compare the cell phenotype subset composition between the two groups and corrected for multiple hypothesis testing using the Bonferroni method.

Reviewer #2:
Remarks to the Author: Sun et al set out to understand early events of Mtb infection with the main question been if resisters have any unique anti-bacterial functional programs relative to latently infected persons.A long outstanding question in the field of TB is understanding the phenomenon of resistors and whether failure to convert TST or IGRA has a biological association with Mtb clearance.They aimed to measure IFNgindependent T cell signatures specific to household exposure, and to determine genes selectively enriched among Mtb-specific T cells derived from resisters compared to controls.This will fill in knowledge gaps about whether resisters have actual protective immunity or failed to convert TST/IGRAs in addition to the known Epi data.They observed a role of Th17 cells and lower polyfunctionality scores in TST-/IGRA relative to LTBI, in addition, they showed important information on T cell quality which is novel.They used other cohorts to confirm this and non-human primates.The manuscript is well-written, experiments were well described as well as the results and conclusions which were precise and concise.The study is important and would have a major impact on the field, however, some of the claims are not confirmed resulting in several limitations.I'm requesting for a major revision due to importance of the paper in the field.
We welcome the positive assessment of our work and the opportunity to address these important critiques.

Major limitations
The study is important and will have a major impact on the field, however these points should be addressed before the paper can be considered for publication in Nature Medicine.1.The authors claim that the observed Th17 phenotype (a known correlate of protection both in human and animal studies); they show is reflective of bacterial burden.However, they have not proven this.I would recommend they measure bacterial burden in the study cohorts to confirm this claim.How do the bacterial burden correlate with the responses observed?
We appreciate this comment but note that, unlike active TB, there are no established methods in the field to quantify bacterial burdens in healthy asymptomatic populations (RSTRs and LTBIs).Our overall thesis is that the T cell signatures that we observe in this study may reflect the final result (bacterial control) of a sequence of events that may have taken place nearly 9 years prior to sample collection.To address this, we extended our findings to collaborative studies of non-human primates, where quantitative bacterial burdens are known.As described above in the reply to Reviewer #1, Comment #15, we now include new data that show positive associations between IFNγ-independent and IL17-expressing CD4 T cells and protection from M.tb in nonhuman primates that have been vaccinated with intravenous BCG (revised Fig 7h and 7i).In addition, we extended our results to a longitudinal human cohort of adolescents with latent M.tb infection that is wellestablished in the field.In Fig. 7a-e, we show that M.tb-specific Th17 T cells are associated with lack of progression to TB disease.This finding is notable because it reveals that the protective signature is not limited to 'resisters' but may also be seen in a subset of individuals with latent TB, thus highlighting the importance of our findings across the spectrum of TB disease.Changes to the text have been detailed in response to Reviewer #1, Comment #2 above.We have also revised the title to be more conservative and emphasize the associative nature of our findings.
Line 2: T cell phenotypes in M. tuberculosis 'resisters' are associated with bacterial control 2. TB has a diverse set of antigens, the authors used only two antigens, ESAT-6 and CFP which are used in IGRAs.The study participants were classified based on IGRAs and TSTs, and persons who were IGRA negative were classified as resistors.IGRAs measure IFNg responses from T cells, thus their observed results that the quality of IFNg response in resistors is low is expected.Is the response observed after ESAT^ and CFP10 stimulation the same as in other TB antigens such as MTB300?I'm recommending that the authors test other TB antigens in the same way as ESAT-6/CFP10 to confirm their results.
This was a consistent critique from two of the three Reviewers.In response, we performed new experiments on RSTR (n=17) and LTBI (n=20) and analyzed T cell phenotypes with flow cytometry after stimulation with M.tb lysate.The findings broadly confirmed our results using ESAT-6/CFP-10 stimulation and have been incorporated into revised Fig. 6.Please see response to Reviewer #1, Comment #5 above for full details.
3. The resistor/LTBI cohort used for targeted sequencing is small.Sample size should be increased to increase the statistical observations made in Figure 2 after index sorting and targeted sequencing.
The targeted sequencing in the discovery phase was meant to be hypothesis generating and followed up in the validation phase using independent methods (flow cytometry and Luminex) and non-overlapping samples (Fig. 1 schema).We have included new targeted transcriptional profiling data on ~16,000 cells derived RSTR (n=16) and LTBI (n=16) after M.tb-lysate stimulation.Please see detailed response to Reviewer #1, Comment #5 above.Overall, we found that the targeted transcriptional profiles were broadly similar among the two groups (new Supp Fig. 7a).Additionally, we have amended the text as follows: In the Supplementary Figures PDF:

Decision Letter, first revision:
Dear Dr. Seshadri, Thank you for your response to the reviewers' comments on your manuscript "T cell phenotypes in M. tuberculosis 'resisters' are associated with bacterial control".We are happy to inform you that if you revise your manuscript appropriately in response to the referees' comments and our editorial requirements your manuscript should be publishable in Nature Immunology.
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In recognition of the time and expertise our reviewers provide to Nature Immunology's editorial process, we would like to formally acknowledge their contribution to the external peer review of your manuscript entitled "T cell phenotypes in M. tuberculosis 'resisters' are associated with bacterial control".For those reviewers who give their assent, we will be publishing their names alongside the published article.Line 712: Typo, please delete "the" before whether.
Reviewer #2 (Remarks to the Author): This is a well-written paper which provides extensive insight into resister control of infection in this highly unique cohort from Uganda.The South African cohort has been leveraged well to validate findings in terms of Th17-like function.
I previously reviewed this manuscript and have carefully checked the authors rebuttal to my comments.They have incorporated all my suggestions and have performed additional experiments to clarify some comments raised by myself and the other reviewers (namely in terms of the stimulation condition used).Responses were thorough and detailed and the manuscript has improved in clarity and scientific impact.
Two points for clarification.Firstly, in the discussion, lines 339-441.I think the addition of 'were' in "Mtb were exposed, but TST-negative..." may be incorrect as the sentence currently doesn't make sense.Secondly, in the methods for select seq you use anti-CD154 incubation for 30 min to prevent downregulation of CD154 but in the T cell proliferation assay you use anti-CD40.Why the difference?
Two points for clarification.Firstly, in the discussion, lines 339-441.I think the addition of 'were' in "Mtb were exposed, but TST-negative..." may be incorrect as the sentence currently doesn't make sense.Secondly, in the methods for select seq you use anti-CD154 incubation for 30 min to prevent downregulation of CD154 but in the T cell proliferation assay you use anti-CD40.Why the difference?
We appreciate reviewers 2 approval of the changes made and new data included in the revised version of our manuscript.Additionally, we thank for the reviewer for pointing out these minor errors/typos: The following have been rectified: • The additional "were" has been removed and now reads as the following: Lines 439-441: "Monoclonal antibodies derived from M.tb exposed, but TST-negative healthcare workers can confer protection against M.tb challenge in mice".
• Regarding the use of "anti-CD154" vs "anti-CD40", anti-CD154 was a typo but refers to the use of the same reagent (anti-CD40).We have now standardized the verbiage used to anti-CD40 throughout the manuscript.

Decision Letter, second revision:
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Fig
Fig. 1a study schema.We analyzed endemic controls with LTBI (n = 19) and matched TST-/IGRA-(n = 17) participants, as well as RSTR (n = 17) and LTBI (n = 20) subjects enrolled through our household contact study.The functional diversity of T cells specific for ESAT-6/CFP-10 or whole M.tb lysate was assessed by intracellular cytokine staining (ICS), targeted transcriptional profiling, and SELECT-Seq.Results were validated experimentally in an independent set of RSTRs (n = 17) and LTBIs (n = 20) using flow cytometry and Luminex.
This is an excellent suggestion.Around the time this manuscript was originally submitted for review, Liu et al. analyzed whole blood transcriptional profiles in a dose-ranging study of IV BCG in rhesus macaques (Liu et al. Cell Reports Medicine 2023, PMID: 37390827).They identified early innate transcriptomic response as immune correlates of protection in this setting.A parallel study by Darrah et al. examined T cell phenotypes in airway and blood(Darrah et al. Cell Host Microbe 2023, PMID: 37267955).This study identified the combined immune feature of frequency of CD4 T cells producing TNF with IFN-γ, frequency of those producing TNF with IL17, and the number of NK cells as immune correlates of protection.
as follows and added the following text to the Methods and Results: Lines 930-939, Fig 7h and 7i legends: (h) Whole blood transcriptional profiles from dose-ranging study of intravenous (IV) BCG of rhesus macaques 57 were used to calculate the Th17 gene module score based on genes identified in Fig.
Lines 788-789, Fig 1 legend: Reported p-values in (d) were adjusted for multiple hypothesis testing using the Bonferroni method.Lines 823-824, Fig 3 legend: Statistical testing was performed using the Wilcoxon rank-sum tests with corrections for multiple-hypothesis testing using the Bonferroni method.Lines 837-838, Fig 4 legend: Statistical testing was performed using the Wilcoxon rank-sum tests with correction for multiple hypothesis testing using the Bonferroni method.Lines 871-873, Fig 5 legend: Statistical significance was determined using Wilcoxon rank-sum tests with correction for multiple hypothesis testing using the Bonferroni method.
When you are ready to submit your revised manuscript, please use the URL below to submit the revised version:We hope to receive your revised manuscript in 7 days, by 24th April.Please let us know if circumstances will delay submission beyond this time.If you have any questions please do not hesitate to contact me..com/niReviewer #1 (Remarks to the Author):Sun et al set out to determine the early events associated with Mtb control and/or clearance.The additional experiments including regulatory T cell measurements on flow cytometry and analyses of published datasets.They also included the Mtb H37RV lysate as an alternative TB antigen which showed similar results to the Esat6/CFP10 antigen confirming the results are not skewed due to IGRA results.They have made efforts to further analyze published datasets, such as the Scriba Plos Pathogens dataset.I think they have covered all the previous comments and have clarified ambiguous points in the manuscript.The manuscript is well-written, the experiments are well described, and I think the results and conclusions match the data shown.I do not have any major comments and have only one minor comment.
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Dear Dr. Seshadri, I am delighted to accept your manuscript entitled "Specific CD4+ T cell phenotypes associate with bacterial control in people who 'resist' infection with Mycobacterium tuberculosis" for publication in an upcoming issue of Nature Immunology.